Species pool and soil properties in mangrove habitats influence the species-immigration process of diazotrophic communities across southern China

ABSTRACT Microbial immigration is an ecological process in natural environments; however, the ecological trade-off mechanisms that govern the balance between species extinction and migration are still lacking. In this study, we investigated the mechanisms underlying the migration of diazotrophic communities from soil to leaves across six natural mangrove habitats in southern China. The results showed that the diazotrophic alpha and beta diversity exhibited significant regional and locational variations. The diazotrophic species pool gradually increased from the leaves to nonrhizosphere soil at each site, exhibiting a vertical distribution pattern. Mantel test analyses suggested that climate factors, particularly mean annual temperature, significantly influenced the structure of the diazotrophic community. The diazotrophic community assembly was mainly governed by dispersal limitation in soil and root samples, whereas dispersal limitation and ecological drift were dominant in leaves. Partial least squares path modeling revealed that the species pool and soil properties, particularly the oxidation-reduction potential and pH, were closely linked to the species-immigration ratio of diazotrophic communities. Our study provides novel insights for understanding the ecological trait diversity patterns and spread pathways of functional microbial communities between below- and aboveground habitats in natural ecosystems. IMPORTANCE Environmental selection plays key roles in microbial transmission. In this study, we have provided a comprehensive framework to elucidate the driving patterns of the ecological trade-offs in diazotrophic communities across large-scale mangrove habitats. Our research revealed that Bradyrhizobium japonicum, Marinobacterium lutimaris, and Agrobacterium tumefaciens were more abundant in root-associated soil than in leaves by internal and external pathways. The nonrhizospheric and rhizospheric soil samples harbored the most core amplicon sequence variants, indicating that these dominant diazotrophs could adapt to broader ecological niches. Correlation analysis indicated that the diversities of the diazotrophic community were regulated by biotic and abiotic factors. Furthermore, this study found a lower species immigration ratio in the soil than in the leaves. Both species pool and soil properties regulate the species-immigration mechanisms of the diazotrophic community. These results suggest that substantial species immigration is a widespread ecological process, leading to alterations in local community diversity across diverse host environments.

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Reviewer #1 (Comments for the Author): This study explored the immigration of diazotrophic communities from soil to leaves across six natural mangrove habitats in China, focusing on the impact of biotic and abiotic factors on functional microbial communities.Using a null model, the author analyzed the assembly processes of diazotrophic communities in various regions.Additionally, the research highlighted the significant role of the species pool and soil properties, such as MAT and pH, in influencing species migration.These findings enhance our understanding of species dispersal dynamics between belowground and aboveground habitats within mangrove ecosystems.Although the study is data-rich and well-written, it lacks adequate detail concerning the categorization of research subjects and the explanation of species migration mechanisms.Moreover, the presentation and verification of results require careful formatting and significance analysis.Comments for revision are provided below.1.Why is there a division of regions into different latitudes?The latitude span here is relatively small (6 degrees), while the longitude has a larger variation (10 degrees).2. Lines 459-461: Is the species pool related to alpha and beta diversity?This relationship lacks detailed explanation and indepth discussion about how alpha and beta diversity influence species pools.3. The significance markers in Fig. S9, such as 'a', 'd', 'f', 'g', 'h', appear confusing.Please review and clarify these results.4. Lines 70-102: The introduction discussing microorganisms with BNF function should be more concise as it does not seem to effectively introduce the topic.5. Please adjust the orientation of the vertical coordinate in Figure 3, for instance, tilting it to 45 degrees to avoid confusion.6.In Figure 4a, are there significant differences in the number of ASVs at different locations?Consider removing the error bars unless significance analysis results are provided.7. Figure 5: Please include significance analysis results.8. Lines 57-63: This text is overly lengthy.Please consider shortening it.9. Lines 67-69: This statement does not adequately express a cause and effect relationship.Please revise for clearer expression.10.Line 123: Replace "no gap is mentioned here" with "Here."11.Lines 266-268: Please provide specific statistical data.12. Lines 351 and 532: Remove the word "all." 13.Line 434: The amplicon sequencing does not characterize active microorganisms, please rephrase it.14. Lines 461-464: Consider splitting the content after "and MAT" into a separate sentence for clarity.15.Line 475: There is a syntax error.Please rewrite this text for clarity.16. Figure S8 is crucial for interpreting the results and should be included as a main figure, displayed together with Figure 4.

Reviewer #2 (Comments for the Author):
This article primarily focuses on the assembly and migration of diazotrophic communities within mangrove habitats, elucidating the impacts of both biotic and abiotic factors on diazotrophic microbial populations.Several issues requiring clarification or improvement are identified within the manuscript: The ecological significance or potential applications of understanding the migration of diazotrophic communities are not adequately addressed in the manuscript.Lines 36-38: Can "species-immigration mechanisms" be regulated, besides being investigated and discovered?Lines 130-132: This study only addresses diazotrophic communities and could not reflect the migration status of the entire microbial community.Lines 150-165: A more detailed description of the methods employed for detecting soil biochemical factor is necessary.Line 165: Parentheses are colored red; please amend to black.Lines 205-206: Which function was used to calculate the "species pool"?Lines 206-208: Reference to the R package is missing.Lines 210-211: Further elaboration is needed on the method used to assess community assembly.Lines 211-212: Can ggplot2 be directly used for conducting linear regression analysis and obtaining relevant results, in addition to being used for plotting linear regression graphs?Lines 212-213: An explanation is required on how Groups I-IV were classified.Lines 255-256: Why does biodiversity increase with increasing geographical distance?What factors drive this?Lines 270-272: Which of the biotic and abiotic factors is more important in driving the assembly of diazotrophic communities?Lines 418-421: The font does not match the rest of the text.The bar plot in Figure 7 lacks a title for the x-axis.

Dear editor,
Thank you very much for your kind letter on April 30, 2024 concerning our manuscript (mSystems00307-24) and for inviting us to submit a revised version of the manuscript.
We have revised the manuscript in accordance with the reviewers' comments and suggestions, and carefully proof-read the manuscript.Please see below for our point-to-point responses to the reviewers' comments and suggestions.
We would like to thank all the reviewers for useful advice, critical comments and valuable suggestions.We greatly appreciate editor's time and excellent guidance to help us to make our manuscript publishable.
If you have any questions, please don't hesitate to contact me.This study explored the immigration of diazotrophic communities from soil to leaves across six natural mangrove habitats in China, focusing on the impact of biotic and abiotic factors on functional microbial communities.Using a null model, the author analyzed the assembly processes of diazotrophic communities in various regions.
Additionally, the research highlighted the significant role of the species pool and soil properties, such as MAT and pH, in influencing species migration.These findings enhance our understanding of species dispersal dynamics between belowground and aboveground habitats within mangrove ecosystems.Although the study is data-rich and well-written, it lacks adequate detail concerning the categorization of research subjects and the explanation of species migration mechanisms.Moreover, the presentation and verification of results require careful formatting and significance analysis.Comments for revision are provided below: 1. Why is there a division of regions into different latitudes?The latitude span here is relatively small (6 degrees), while the longitude has a larger variation (10 degrees).
Response：Many thanks.Mangrove forests in China are mainly distributed along the southern and southeastern coastal regions, including provinces such as Guangdong, Guangxi, Fujian, and Hainan (1).Generally, an increase in temperature and precipitation is expected in regions with lower latitudes, whereas a decrease in these parameters is expected in northward transplantation (climate cooling) at northern latitudes along the coastal areas (2).Climate is an important environmental determinant of species distribution, and climate change has significant impacts on biodiversity, including species distribution and interspecific interactions (3).In this study, we utilized longitude and latitude as geographical distances to investigate their effects on biodiversity.We feel to mark the samples based on latitude to highlight the differences in the distribution of diazotrophic communities between northern-southern regions of mangrove forests, facilitating a better comprehension for readers.However, if you believe in using longitude to mark the samples is more appropriate, we can make that adjustment.
The above reference mentioned had been listed as the following: 3. The significance markers in Fig. S9, such as 'a', 'd', 'f', 'g', 'h', appear confusing.
Please review and clarify these results.
Response：Thank you very much for your good comments and numerous valuable suggestions.We have revised the legend of Fig. S10 (previous Fig. S9) to explain the meaning of the different letters (significant difference at P < 0.05.) and made corrections to Fig. S10 to avoid confusion.Response：Many thanks for your good comments and numerous valuable suggestions.
We have revised the sentences as the following described: "To explore transmission processes at both the individual plant and ecosystem levels, we need to study endophyte colonization routes and dispersal modes, for example, microbial communities assemble and immigration mechanisms between belowground (i.e., soil and root) and aboveground (i.e., leaf) host environment."(Clean Version line: L79-82) 9. Lines 67-69: This statement does not adequately express a cause and effect relationship.Please revise for clearer expression.
Response：Thank you very much for your valuable comments.We have revised the sentences as the following described: "Mangrove ecosystems are rich in organic carbon and nitrogen, making them ideal habitats for microbial communities, it is necessary to evaluate the migration or transmission processes between below-and aboveground habitats in the functional microbiome."(Clean Version line: L86-89) 10.Line 123: Replace "no gap is mentioned here" with "Here." Response：Many thanks.According to the comments, we have revised the sentences as the following described: "Here, we collected sediment, rhizosphere soil, root, and leaf samples from the popular species Kandelia obovata from coastal mangroves across six natural coastal mangrove wetlands in China, spanning Response ： Thank you very much for your important comments and valuable suggestions.We have added specific statistical results in the sentences as the flowing described: "Soil texture, including sand, silt, and clay (R = 0.217-0.426,P < 0.001), was more important for the root-associated diazotrophic community than for the leaves.Response：Thank you very much for your good comments and numerous valuable suggestions.We have revised the sentence as the flowing described: "Bradyrhizobium japonicum, Marinobacterium lutimaris, and Agrobacterium tumefaciens were most abundant species, indicating their broad ecological niche in interspecies competition (Fig. S1)." (Clean Version line:447-449) 14. Lines 461-464: Consider splitting the content after "and MAT" into a separate sentence for clarity.
Response：Many thanks.According to the comments, we have revised the sentence as the flowing described: "In our study, the variability in the diazotrophic community structure was strongly correlated with MAT, salinity, PTC, and TOC.Wherein MAT (R = 0.318-0.432,P < 0.001) was confirmed to be the most crucial driving factor (Fig.  The ecological significance or potential applications of understanding the migration of diazotrophic communities are not adequately addressed in the manuscript.
Response ： Species-immigration mechanisms are regulated by biodiversity and environmental conditions.For instance, habitat destruction, environmental pollution and anthropogenic global changes (e.g.climate change) pose severe challenges to local habitat biodiversity (1).Habitat loss has pervasive and disruptive impacts on biodiversity, causing exceptionally high for the rates of species extinctions (2,3).In our study, pearson's correlation analysis and least squares path modelling (PLS-PM) show that species pool and soil properties can significantly influence the species-immigration mechanisms.However, we are not available to utilize specific isotope-labeled compounds (such as 13 C, 15 N, etc.) to track their metabolism and transformation processes for diazotrophic microbial communities within the rhizosphere soil-plant system.In this study, we used the conventional ecological methods such as species composition, diversity analysis, and community assembly, combined with the model was described by Hillebrand and Sun et al (3,4), assessing the migration mechanisms of diazotrophic microbial communities within the rhizosphere mangrove soil-plant systems.From an ecological perspective, this study offers new insights for understanding the ecological trait diversity patterns and spread pathways of functional microbial communities between below and aboveground habitats in natural ecosystems.
The above reference mentioned had been listed as the following:

I
look forward to hearing from you soon.With the best kindly regards; Nanli Prof. Nan Li Key Laboratory of Climate, Resources and Environment in Continental Shelf Sea and Deep Sea of Department of Education of Guangdong Province, Department of Oceanography, Key Laboratory for Coastal Ocean Variation and Disaster Prediction, College of Ocean and Meteorology, Guangdong Ocean University, Zhanjiang 524088, People's Republic of China.

Fig. S10
Fig. S10 The variation in immigration mechanisms of diazotrophic communities

Fig. 5 .
Fig. 5. Variations of the immigration process of diazotrophic communities

However, SO 4 2 -
(R = 0.322, P < 0.01) was the main driving factor affecting the diazotrophic community in the rhizosphere soil."(Clean Version line:273-276) 12. Lines 351 and 532: Remove the word "all."Response： Many thanks.We have revised the manuscript, please check that in the new version.(Clean Version line:366 and 554) 13.Line 434: The amplicon sequencing does not characterize active microorganisms, please rephrase it.

Fig. 4 .
Fig. 4. Distribution patterns of the shared and unique ASVs in different groups.